Comparison of Hydroxyapatite/Poly(lactide-co-glycolide) and Hydroxyapatite/Polyethyleneimine Composite Scaffolds in Bone Regeneration of Swine Mandibular Critical Size Defects: In Vivo Study.

Reconstruction of jaw bone defects present a significant problem because of specific aesthetic and functional requirements. Although widely used, the transplantation of standard autograft and allograft materials is still associated with significant constraints. Composite scaffolds, combining advantages of biodegradable polymers with bioceramics, have potential to overcome limitations of standard grafts. Polyethyleneimine could be an interesting novel biocompatible polymer for scaffold construction due to its biocompatibility and chemical structure. To date, there have been no in vivo studies assessing biological properties of hydroxyapatite bioceramics scaffold modified with polyethyleneimine. The aim of this study was to evaluate in vivo effects of composite scaffolds of hydroxyapatite ceramics and poly(lactide-co-glycolide) and novel polyethyleneimine on bone repair in swine's mandibular defects, and to compare them to conventional bone allograft (BioOss). Scaffolds were prepared using the method of polymer foam template in three steps. Pigs, 3 months old, were used and defects were made in the canine, premolar, and molar area of their mandibles. Four months following the surgical procedure, the bone was analyzed using radiological, histological, and gene expression techniques. Hydroxyapatite ceramics/polyethyleneimine composite scaffold demonstrated improved biological behavior compared to conventional allograft in treatment of swine's mandibular defects, in terms of bone density and bone tissue histological characteristics.

Biocompatibility Study of a New Dental Cement Based on Hydroxyapatite and Calcium Silicates: Focus on Liver, Kidney, and Spleen Tissue Effects.

The effects of a new material based on hydroxyapatite and calcium silicates, named ALBO-MPCA, were investigated on the liver, kidney and spleen. The material was administrated orally for 120 days in an in vivo model in Wistar rats, and untreated animals served as a control. Hematological and biochemical blood parameters were analyzed. Qualitative histological analysis of tissues, change in mitotic activity of cells, and histological characteristics was conducted, as well as quantitative stereological analysis of parenchymal cells, blood sinusoids, and connective tissues. Additionally, the protein expressions of Ki67 and CD68 markers were evaluated. Histological analysis revealed no pathological changes after the tested period. It showed the preservation of the architecture of blood sinusoids and epithelial cells and the presence of mitosis. Additionally, the significantly increased number of the Ki67 in the presence of ALBO-MPCA confirmed the proliferative effect of the material noticed by stereological analysis, while immunoreactive CD68 positive cells did not differ between groups. The study showed non-toxicity of the tested material based on the effects on the hematological, biochemical, and observed histological parameters; in addition, it showed evidence of its biocompatibility. These results could be the basis for further steps toward the application of tested materials in endodontics.

Nanostructured endodontic materials mixed with different radiocontrast agents-biocompatibility study.

The aim of this study was to investigate the biocompatibility of nanostructured materials based on highly active calcium silicates mixed with different radiocontrast agents in comparison to MTA+ using in vitro and in vivo model. Morphology of materials' samples was analyzed using SEM while the phase compositions were identified by XRD. pH values of materials' suspensions were conducted by pH-meter. The cytotoxicity of materials' solutions was tested by MTT test (100, 50, 25 and 12.5 mg/ml). LDH and 3H-thymidine assay were utilized for biocompatibility investigations of materials' eluates (24 h, 7 day and 21 day). Eighteen Guinea pigs were used for intramuscular implantation, as teflon tubes with freshly prepared materials were placed into intramuscular pockets. All samples were composed of round and needle-like particles equally distributed with Ca/Si ratio ~2.7 at%, with the presence of hydrated calcium silicate phases. The pH values of ALBO-MPCA1 and ALBO-MPCA2 were high alkaline, while in case of MTA+ they were lower and continuously declined (p < 0.05). Investigated materials didn't exhibit dose-dependent effect on metabolic activity of L929 cells (p > 0.05). Significant differences in the percentage of cytotoxicity between diluted and undiluted extracts between all tested materials after 24 h and 7 day were noticed (p < 0.05). Increase in L929 cells proliferation was noticed in case of undiluted eluates of ALBO-MPCA1 and ALBO-MPCA2 after 7 day (p < 0.05). There were no statistically significant differences in the intensity of inflammatory response between investigated materials and control group after 60 day (p > 0.05). Evaluation of biocompatibility of both ALBO-MPCA1 and ALBO-MPCA2 indicate their potential clinical use.

Nanodesigned coatings obtained by plasma electrolytic oxidation of titanium implant and their cytotoxicity.

The titanium implant was treated with plasma electrolytic oxidation and subsequent ionic exchange and thermal treatment in order to obtain bioactive layer consisting of titanium oxide, calcium and sodium titanates and hydroxyapatite, as confirmed by X-ray diffraction (XRD). Scanning electron microscopy (SEM) revealed that the given method, besides corresponding phase composition, enables suitable nanotopology for cell attachment and proliferation. Cytotoxicity investigations by MTT, LDH and propidium iodide assays and light microscopy showed that these coatings were not toxic to L929 cells.

Toxicological Profile of Nanostructured Bone Substitute Based on Hydroxyapatite and Poly(lactide-co-glycolide) after Subchronic Oral Exposure of Rats.

Novel three-dimensional (3D) nanohydroxyapatite-PLGA scaffolds with high porosity was developed to better mimic mineral component and microstructure of natural bone. To perform a final assessment of this nanomaterial as a potential bone substitute, its toxicological profile was particularly investigated. Therefore, we performed a comet assay on human monocytes for in vitro genotoxicity investigation, and the systemic subchronic toxicity investigation on rats being per oral feed with exactly administrated extract quantities of the nano calcium hydroxyapatite covered with tiny layers of PLGA (ALBO-OS) for 120 days. Histological and stereological parameters of the liver, kidney, and spleen tissue were analyzed. Comet assay revealed low genotoxic potential, while histological analysis and stereological investigation revealed no significant changes in exposed animals when compared to controls, although the volume density of blood sinusoids and connective tissue, as well as numerical density and number of mitosis were slightly increased. Additionally, despite the significantly increased average number of the Ki67 and slightly increased number of CD68 positive cells in the presence of ALBO-OS, immunoreactive cells proliferation was almost neglected. Blood analyses showed that all of the blood parameters in rats fed with extract nanomaterial are comparable with corresponding parameters of no feed rats, taken as blind probe. This study contributes to the toxicological profiling of ALBO-OS scaffold for potential future application in bone tissue engineering.

Developing a novel resorptive hydroxyapatite-based bone substitute for over-critical size defect reconstruction: physicochemical and biological characterization and proof of concept in segmental rabbit's ulna reconstruction.

The aim of this study was to develop novel hydroxyapatite (HAP)-based bioactive bone replacement materials for segmental osteotomy reconstruction. Customized three-dimensional (3D) bone construct was manufactured from nanohydroxyapatite (nHAP) with poly(lactide-co-glycolide) (PLGA) coating using 3D models derived from the computed tomography (CT) scanning of the rabbit's ulna and gradient 3D printing of the bone substitute mimicking the anatomical shape of the natural bone defect. Engineered construct revealed adequate micro-architectural design for successful bone regeneration having a total porosity of 64% and an average pore size of 256 µm. Radiography and micro-CT analysis depicted new bone apposition through the whole length of the reconstructed ulna with a small area of non-resorbed construct in the central area of defect. Histological analysis revealed new bone formation with both endochondral and endesmal type of ossification. Immunohistochemistry analysis depicted the presence of bone formation indicators - bone morphogenetic protein (BMP), osteocalcin (OCN) and osteopontin (OPN) within newly formed bone. Manufactured personalized construct acts as a "smart" responsive biomaterial capable of modulating the functionality and potential for the personalized bone reconstruction on a clinically relevant length scale.

Biocompatibility Investigation of New Endodontic Materials Based on Nanosynthesized Calcium Silicates Combined with Different Radiopacifiers.

INTRODUCTION: The aim of this article was to analyze biocompatibility and bioactivity of new endodontic materials on the basis of nanosynthesized calcium silicates (ALBO-MPCA1 and ALBO-MPCA2) combined with different radiopacifiers in comparison with MTA+. METHODS: Morphology of the samples was studied by scanning electron microscopy, and the pH and ion release analysis were also assessed. Biocompatibility of materials' eluates (24-hour, 7-day, and 21-day) was conducted by using MTT test. Twelve New Zealand white rabbits were used for intraosseous implantation. Four calvarial defects per animal were created and filled with freshly prepared investigated materials. RESULTS: Samples mostly consisted of agglomerates built up from nanoparticles, preferably spherical and rod-like. There was no significant difference among pH values of materials' eluates after 24 hours (P > .05). The amount of calcium and aluminum ion release decreased, whereas the amount of magnesium and bismuth (ALBO-MPCA1, MTA+) and barium (ALBO-MPCA2) increased during 21-day period. The metabolic activity of cells increased after the extraction time, except in case of undiluted elutes of ALBO-MPCA2 and ALBO-MPCA1 (21-day). Histologic analysis of the samples revealed newly formed bone tissue with moderate inflammation for all investigated materials, which subsided during 90-day period to mild. Both MTA+ and ALBO-MPCA1 were in direct contact with the newly formed bone tissue. After 90 days, statistically significant difference in hard tissue formation was observed in comparison of MTA+ and ALBO-MPCA1 with control group (P < .05). CONCLUSIONS: Experimental materials ALBO-MPCA1 and ALBO-MPCA2 possess both biocompatibility and bioactivity. Because ALBO-MPCA1 provokes favorable biological response, it is especially good candidate for further clinical investigations.

Extraordinary biological properties of a new calcium hydroxyapatite/poly(lactide-co-glycolide)-based scaffold confirmed by in vivo investigation.

This study examined the potential of a new porous calcium hydroxyapatite scaffold covered with poly (lactide-co-glycolide) (PLGA) as a bone substitute, identifying its advantages over Geistlich Bio-Oss®, considered the gold standard, in in vivo biofunctionality investigations. Structural and morphological properties of the new scaffold were analyzed by scanning electron and atomic force microscopy. The biofunctionality assays were performed on New Zealand white rabbits using new scaffold for filling full-thickness defects of critical size. The evaluated parameters were: the presence of macrophages, giant cells, monoocytes, plasma cells, granulocytes, neoangiogenesis, fibroplasia, and the percentage of mineralization. Parallel biofunctionality assays were performed using Geistlich Bio-Oss®. The appearance of bone defects 12 weeks after the new scaffold implantation showed the presence of a small number of typical immune response cells. Furthermore, significantly reduced number of capillary buds, low intensity of fibroplasia and high degree of mineralization in a lamellar pattern indicated that the inflammation process has been almost completely overcome and that the new bone formed was in the final phase of remodeling. All biofunctionality assays proved the new scaffold's suitability as a bone substitute for applications in maxillofacial surgery. It showed numerous biological advantages over Geistlich Bio-Oss® which was reflected mainly as a lower number of giant cells surrounding implanted material and higher degree of mineralization in new formed bone.

Cytotoxicity investigation of a new hydroxyapatite scaffold with improved structural design.

Introduction: Biodegradable porous scaffolds are found to be very promising bone substitutes, acting as a temporary physical support to guide new tissue regeneration, until the entire scaffold is totally degraded and replaced by the new tissue. Objective: The aim of this study was to investigate cytotoxicity of a synthesized calcium hydroxyapatitebased scaffold, named ALBO-OS, with high porosity and optimal topology. Methods: The ALBO-OS scaffold was synthesized by the method of polymer foam template. The analysis of pore geometry and scaffold walls' topography was made by scanning electron microscope (SEM). The biological investigations assumed the examinations of ALBO-OS cytotoxicity to mouse L929 fibroblasts, using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor (MTT) and lactate dehydrogenase (LDH) tests and inverse phase microscopy. Results: The SEM analysis showed high porosity with fair pore distribution and interesting morphology from the biological standpoint. The biological investigations showed that the material is not cytotoxic to L929 cells. Comparison of ALBO-OS with Bio-Oss, as the global gold standard as a bone substitute, showed similar results in MTT test, while LDH test showed significantly higher rate of cell multiplication with ALBO-OS. Conclusion: The scaffold design from the aspect of pore size, distribution, and topology seems to be very convenient for cell adhesion and occupation, which makes it a promising material as a bone substitute. The results of biological assays proved that ALBO-OS is not cytotoxic for L929 fibroblasts. In comparison with Bio-Oss, similar or even better results were obtained.

Three-dimensional total synchronous luminescence spectroscopy criteria for discrimination between normal and malignant breast tissues.

Specimens of malignant and normal female human breast tissues were analyzed after surgery by means of synchronous luminescence spectroscopy. Measurements were performed in the ranges of excitation wavelengths from 330 to 650 nm and synchronous wavelengths from 30 to 120 nm to obtain ordinary and first derivative three-dimensional total synchronous luminescence spectra (3d-TSLS) of each specimen. Arithmetic mean of these spectra has been calculated for normal and malignant specimens and analyzed to establish criteria for tissue differentiation. Spectral domain volumes (volumes below luminescence intensity surface) and mean spectral slopes have been calculated and also analyzed as tissue discrimination criteria. The obtained results are discussed in view of the possible relevance of synchronous luminescence spectroscopy in discrimination between normal and malignant breast tissue.

Effects of novel hydroxyapatite-based 3D biomaterials on proliferation and osteoblastic differentiation of mesenchymal stem cells.

The aim of this study was to examine the differential capacity of isolated dental pulp stem cells (SHED) cultured onto four different scaffold materials. The differential potential of isolated SHED was examined on the following scaffolds: porous hydroxyapatite (pHAP) alone or combined with three polymers [polylactic-co-glycolic acid (PLGA), alginate, and ethylene vinylacetate / ethylene vinylversatate (EVA/EVV)]. SHED were isolated by "outgrowth" method and characterized by the flow cytometry. Viability of cells grown with scaffolds was assessed by MTT and LDH assays. No significant cytotoxic effect of any of the tested materials was shown. Staining with alizarin red and estimated alkaline phosphatase activity to identify differentiation, demonstrated osteoblastic phenotype of SHED and newly deposited and mineralized extra cellular matrix (ECM) in presence of all tested scaffolds. The developed ECM seen at scanning electronic micrographs additionally confirmed the osteogenic differentiation and biocompatibility between cells and materials. In summary, all studied biomaterials are suitable carriers for proliferation and osteoblastic differentiation of dental pulp mesenchymal stem cells in vitro.

Addition of a Fluoride-containing Radiopacifier Improves Micromechanical and Biological Characteristics of Modified Calcium Silicate Cements.

INTRODUCTION: Calcium silicate cements (CSCs) with the addition of nanohydroxyapatite and calcium carbonate play a critical role in dental applications. To further improve their properties, particularly radiopacity and biointeractivity, the fluoride-containing radiopacifier ytterbium trifluoride (YbF3) was added to their composition, and biological and mechanical characteristics were evaluated. METHODS: YbF3 was added to 3 different CSCs: cement I (CSC + calcium carbonate), cement II (CSC + nanohydroxyapatite), and Portland cement. Material characterization encompassed measurements of pH, calcium, ytterbium, and fluoride ion release; radiopacity; setting time; porosity; microindentation properties; wettability; and Fourier transform infrared spectroscopic, x-ray diffraction, and scanning electron microscopic analyses. Osteoblast- and osteoclast-like cells were grown on the materials' surface to evaluate their adherence. RESULTS: The addition of calcium carbonate, nanohydroxyapatite, and 30 wt% of YbF3 improved radiopacity and the setting time of experimental cements. The pH values did not differ among the groups. The greatest ytterbium and fluoride releases occurred in the Portland cement + YbF3 group. Combined x-ray diffraction and Fourier transform infrared spectroscopic analysis showed the presence of calcium hydroxide and calcium silicate hydrates. In addition, the presence of calcium ytterbium fluoride and ytterbium oxide proved that YbF3 reacted with cement compounds. Wettability of cement I + YbF3 was superior to other formulations, but its porosity and microindentation properties were weaker than in the Portland cement + YbF3 mixture. Cement II + YbF3 presented micromechanical indentation and porosity characteristics similar to the Portland-based cement formulation. Osteoclast- and osteoblast-like cells adhered to the cements' surfaces without alteration of the cell structural integrity. CONCLUSIONS: YbF3-containing CSCs with nanostructured hydroxyapatite and calcium carbonate are well suited for dental application.

The efficacy of hydrothermally obtained carbonated hydroxyapatite in healing alveolar bone defects in rats with or without corticosteroid treatment.

BACKGROUND/AIM: Autogenous bone grafting has been the gold standard in clinical cases when bone grafts are required for bone defects in dentistry. The study was undertaken to evaluate multilevel designed carbonated hydroxyapatite (CHA) obtained by hydrothermal method, as a bone substitute in healing bone defects with or without corticosteroid treatment in rats as assessed by histopathologic methods. METHODS: Bone defects were created in the alveolar bone by teeth extraction in 12 rats. The animals were initially divided into two groups. The experimental group was pretreated with corticosteroids: methylprednisolone and dexamethasone, intramuscularly, while the control group was without therapy. Posterior teeth extraction had been performed after the corticosteroid therapy. The extraction defects were fulfilled with hydroxyapatite with bimodal particle sizes in the range of 50-250 µm and the sample from postextocactional defect of the alveolar bone was analyzed pathohystologically. RESULTS: The histopatological investigations confirmed the biologic properties of the applied material. The evident growth of new bone in the alveolar ridge was clearly noticed in both groups of rats. Carbonated HA obtained by hydrothermal method promoted bone formation in the preformed defects, confirming its efficacy for usage in bone defects. Complete resorption of the material's particles took place after 25 weeks. CONCLUSION: Hydroxyapatite completely meets the clinical requirements for a bone substitute material. Due to its microstructure, complete resorption took place during the observation period of the study. Corticosteroid treatment did not significantly affect new bone formation in the region of postextractional defects.

Use of mineral trioxide aggregate in the treatement of traumatized teeth in children--two case reports.

INTRODUCTION: Dental injuries in immature permanent teeth often result in endodontic complications. Apexification technique using calcium hydroxide is associated with certain flaws, such as long treatment time, the possibility of tooth fracture and incomplete calcification. The use of an apical plug employing mineral trioxide aggregate (MTA) is an alternative treatment option. CASE REPORT: We reported the successful treatment of 4 maxillary incisors (in a 7-year-old boy and a 10-year-old girl) with open apices and periapical lesions. Apical portions of the canals were filled with MTA plugs in both cases. Coronal parts of the root canals were filled with gutta-percha and sealer. Clinical findings were clear 6 months after the definite obturation with no pathological changes on the radiographs in both cases. CONCLUSION: The use of MTA for apical plugging appears to be a valid treatment option in traumatized immature teeth with endodontic complications.

The protection of cells from nitric oxide-mediated apoptotic death by mechanochemically synthesized fullerene (C(60)) nanoparticles.

The influence of fullerene (C(60)) nanoparticles on the cytotoxicity of a highly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticles were prepared by mechanochemically assisted complexation with anionic surfactant sodium dodecyl sulfate, macrocyclic oligosaccharide gamma-cyclodextrin or the copolymer ethylene vinyl acetate-ethylene vinyl versatate. C(60) nanoparticles were characterized by UV-vis and atomic force microscopy. While readily internalized by mouse L929 fibroblasts, C(60) nanoparticles were not cytotoxic. Moreover, they partially protected L929 cells from the cytotoxic effect of NO-releasing compounds sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholino-sydnonimine (SIN-1). C(60) nanoparticles reduced SNP-induced apoptotic cell death by preventing mitochondrial depolarization, caspase activation, cell membrane phosphatidylserine exposure and DNA fragmentation. The protective action of C(60) nanoparticles was not exerted via direct interaction with NO, but through neutralization of mitochondria-produced superoxide radical in NO-treated cells, as demonstrated by using different redox-sensitive reporter fluorochromes. These data suggest that C(60) complexes with appropriate host molecules might be plausible candidates for preventing NO-mediated cell injury in inflammatory/autoimmune disorders.